Interpretive Summary: Initial research indicated that white locoweed had increased production of an alkaloid (swainsonine) when plant roots received higher levels of nitrogen from one strain of Rhizobium nitrogen-fixing bacteria. Here, we examined white locoweed and three varieties of woolly locoweed secondary metabolite (swainsonine), primary physiology (photosynthetic activity, pigment concentrations), and growth (shoot and root biomass) dose responses to supplemented nitrogen (ammonium nitrate) fertilizer. Relative to plants grown at the lowest nitrogen dose, plants receiving a 20x greater nitrogen dose had modest increases in leaf photosynthetic rate and pigment concentration increases, as well as shoot biomass. In contrast, plants receiving a 100x greater nitrogen dose had large increases in leaf photosynthetic rate and pigment concentrations, as well as shoot and root biomass. There were differences among the four locoweeds in the degree of physiological and growth responses. Yet, white locoweed and one woolly locoweed variety had swainsonine decreases in plants receiving higher nitrogen doses, one woolly locoweed variety that had no change in swainsonine levels, and a barely detectable swainsonine woolly locoweed variety had small but transient increases in swainsonine levels. Our results indicate that locoweeds were absorbing and using supplemented nitrogen to augment primary physiological activity (photosynthesis and pigment production) and subsequent growth, and allocated more to promoting additional shoot growth over root growth. However, the fungal endophyte that actually produces swainsonine within locoweeds had taxon-specific responses to nitrogen supplementation (increasing nitrogen provided to plants) that generally was not positive. Our nitrogen supplementation study relates to efforts to understand how environmental factors influence locoweed swainsonine production. Livestock poisoning (‘locoism’) from locoweed consumption has been an economic problem in the western U.S. for decades. Nitrogen supplementation had at best inconsistent effects on swainsonine production across our test locoweeds. However, we observed increases in swainsonine levels over time independent on N supplementation for all three woolly locoweed varieties, and decreases over time for white locoweed. Thus, future efforts will examine whether factors related to time (photoperiod, temperature) might influence seasonal locoweed swainsonine production.

Technical Abstract:
• Premise of the study. Plant communities may be influenced by toxic secondary metabolites or enhanced plant growth from plant-symbiont interactions. The C:N hypothesis predicts that C or N constrains plant secondary metabolite production, but does not consider compounds produced by plant symbionts. Locoweeds are legumes that can have fungal endophyte alkaloid (swainsonine- SWA) production which causes livestock poisoning. We study four locoweed taxa to test whether average SWA concentrations influence SWA positive dose responses to N fertilizer.
• Methods. We measured locoweed leaf SWA, pigment concentrations and photosynthetic activity, and plant biomass dose responses to nitrogen supplementation for three months in two greenhouse experiments.
• Key results. Leaf photosynthesis and pigment concentrations, and plant biomass had positive, unsaturated dose responses across tested N doses. While N enhanced primary growth, two moderate SWA taxa (Astragalus mollissimus var. bigelovii and Oxytropis sericea) had negative SWA dose responses to increasing N, the high SWA taxon (A. moll. var. mollissimus) had no SWA change, and the very low SWA taxon (A. moll. var. matthewsii) had a transient positive dose response.
• Conclusions. Supplemented N led to positive dose responses for plant biomass and leaf photosynthesis and pigments, but SWA dose responses differed across locoweed taxa and time. At N levels that enhanced plant growth and reduced antioxidant protective systems, fungal endophyte alkaloid production was not strongly influenced. Production of SWA may be more strongly influenced by factors other than C:N supply (e.g., seasonality, plant age) in the locoweed-endophyte-Rhizobium complex.